Anterograde axonal tracing with the subunit B of cholera toxin: A highly sensitive immunohistochemical protocol for revealing fine axonal morphology in adult and neonatal brains
Anterograde axonal tracing with the subunit B of cholera toxin: A highly sensitive immunohistochemical protocol for revealing fine axonal morphology in adult and neonatal brains
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DOI：
10.1016/0165-0270(95)00155-7
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发表时间：
1996-03-01
影响因子：
3
通讯作者：
Sur, M
中科院分区：
文献类型：
--
作者：
Angelucci, A;Clasca, F;Sur, M
我们报告了一种改进的免疫组织化学方案，用于揭示霍乱毒素 (CTB) 亚基 B 的顺行轴突运输，该方案对轴突和末端进行了非常详细的染色，以便可以长距离跟踪单个轴突并完整重建其轴突。我们的修改主要通过增加一抗在组织中的渗透来提高染色质量。该方案可以修改为允许在交替切片中与与辣根过氧化物酶 (WGA-HRP) 缀合的小麦胚芽凝集素 (WGA-HRP) 的四甲基联苯胺 (TMB) 组织化学染色组合。使用该方案，我们在两个实验范例下测试了 CTB 作为顺行示踪剂的性能，这使得其他顺行示踪剂不太敏感或不可靠：(1) 注射到玻璃体后标记整个视网膜离断投影到大脑(2) 标记出生后早期哺乳动物的皮质和丘脑中正在发育的突起。与用于标记这些相同投影的其他示踪剂（菜豆白细胞凝集素、葡聚糖罗丹明、生物素化葡聚糖、游离 WGA 或 WGA-HRP）进行了定性比较。从这些观察结果可以清楚地看出，通过我们的方案可视化的 CTB 提供了更敏感的视网膜离断投影以及新生儿前脑轴突连接的顺行标记。
We report an improved immunohistochemical protocol for revealing anterograde axonal transport of the subunit B of cholera toxin (CTB) which stains axons and terminals in great detail, so that single axons can be followed over long distances and their arbors reconstructed in their entirety. Our modifications enhance the quality of staining mainly by increasing the penetration of the primary antibody in the tissue. The protocol can be modified to allow combination in alternate sections with tetramethylbenzidine (TMB) histochemical staining of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP).Using this protocol, we tested the performance of CTB as an anterograde tracer under two experimental paradigms which render other anterograde tracers less sensitive or unreliable: (1) labeling the entire retinofugal projection to the brain after injections into the vitreal chamber of the eye, and (2) labeling developing projections in the cortex and thalamus of early postnatal mammals. Qualitative comparisons were made with other tracers (Phaseolus vulgaris leucoagglutinin, dextran rhodamine, biotinylated dextran, free WGA, or WGA-HRP) that were used to label these same projections. From these observations it is clear that CTB, visualized with our protocol, provides more sensitive anterograde labeling of retinofugal projections as well as of axonal connections in the neonatal forebrain.